Clearing agents for histological tissue



Patented July 14, 1953 ICE CLEARING AGENTS FOR ,VHISITOLOGICAL TISSUE Andrs Ferrari, Jr., Uniondale, N.'Y.,.assignor to Technicon Chemical Company, Inc., N. Y., a corporation of New York; I

New York,

No Drawing Application August 29,1950,

Serial No. 182,145

2 Claims.

This invention relates to the preparation of histological tissue specimens for microscope examination and, more particularly to clearing agents employed in treating the tissue in the course of such preparation.

The preparation of tissue to enable the microscopic examination thereof involves a series of treatments of the tissue prior to the cutting of the sections from the tissue specimens for the staining and mounting of the sections on the microscope slides. More particularly, in the preparation of the tissue it is necessary to immerse the tissue successively in a series of liquid agents for certain lengths of time, first to fix the tissue, then to wash the same for removing the fixative, then to dehydrate the tissue, usually by immersion of the tissue successively in alcohols or other dehydration agents, then to immerse the tissue in a clearing agent, and thereafter to infiltrate the tissue with an infiltration agent such as, for example, parafiin, celloidin, etc. After the tissue is thus treated, it is cut into sections of the desired thickness; then the parafiin or other infiltration medium is removed from said sections usually by a solvent, after which the sections are stained and mounted on the slides.

As pathologists are well aware, the purpose of treating tissue with a clearing agent before infiltrating the tissue with parafiin is to eliminate from the tissue all liquid which, if not removed, would decrease the refractive index of the tissue and would interfere with the thorough infiltration of the paraffin into the tissue resulting in poorly cut tissue sections. It is of the utmost importance, therefore, that a tissue-clearing agent be capable of removing all such liquid from the tissue and have a high degree of solvency for, or miscibility with, parafiin so as to facilitate the infiltration of the paraflin into the tissue. It is also very important that the clearing agent be of such character that it does not harden the tissue and does not impair the cytological structures of the tissue. Further, among other things, it is desirable that the clearing agent be non-inflammable. The clearing agent of the present invention possesses these and other desirable characteristics and advantages. as will hereinafter more fully appear, and is moreover less expensive than the clearing agent heretofore preferred by the pathologist.

Heretofore, the clearing agent usually preferred by the pathologist consisted of butyl acetate. This substance, although fairly satisfactory in certain respects, especially in that it does not harden and distort the tissue, is not satisfactory in other respects of considerable importance, more especially in that butyl acetate is not as good a solvent for paraffin as maybe desired, has a pungent odor, and is rather expensive.

The primary object of the present invention is the provision of an improved clearing agent in the respect that it possesses the desirable properties of butyl acetate but has in addition a consider ably greater solvent action on fats and waxes and has a much more pleasant odor than butyl acetate.

Pursuant to the present invention, the clearing agent comprises N. butyl acetate, ethylene dichloride and carbon tetrachloride. The function of the ethylene dichloride in the composition of the present clearing agent is to impart to the latter the desired degree of solvency for fats and waxes, which the composition would otherwise not possess in view of the considerably lower degree of solvency of butyl acetate. The function of the carbon tetrachloride is to raise the flash point of the composition so that the clearing agent is non-inflammable.

The following is an example of the presently preferred composition of the clearing agent of the present invention:

N. butyl acetate percent 50.0 Ethylene dichloride do 37.5= Carbon tetrachloride do 12.5

properties of the composition pursuant to the primary object of the invention, as will be apparent to those skilled in the art and in the light of the presence disclosure.

It will be readily understood, of course, that in using the clearing agent of the present invention, the tissue is immersed therein after the dehydration treatment and before the paraflin infiltration process. Tissue blocks comprising tissue which has been treated with the clearing agent of this invention have greatly improved cutting qualities, the cytological structures of the tissues are eifectively preserved, and the appearance of the tissue sections is considerably improved.

It will be understood that various changes may be made in the clearing agent of the'present invention within the skill of the art, without departing from the underlying idea or principles of nliy invention within the scope of the appended c aims.

about theaproportions, by. volume of r.5( )%,.-0f; N.

butyl acetate, ,37.5%"ofethylene dichloride,.,and 12.5% of carbon tetrachloride.

2. A clearing agent for use in the preparation"? of histological tissue for microscopicexamination consisting essentially of N. butyl acetate; ethylene? dichloride and carbon tetrachloride, in which the amount by volume of N. butyl acetatezisapproxiea mately equal to the amountstofcbothithe:ethylenet dichloride and carbon tetrachloride, the amount Number of ethylene dichloride exceeds thelamountioflcare bon tetrachloride and the amount of carbon-i.tet+r-- rachloride is sufficient to render the clearing agent substantially non-inflammable.

v ANDRES FERRARI, JR.

References Cited in the file of this patent UNITED-. STATES PATENTS Number Name Date 1,307,562 Metz et a1 June 24, 1919 1,434,465 Webb Nov. 7, 1922 1,464,170 Carroll Aug. 7, 1923 1,915,163 Morrison et al June 20, 1933 2,393,580 Weiskopf Jan. 22, 1946 FOREIGN PATENTS Country Date 46.1,034L. France Dec. 1'7, 1913 390,867 Great Britain Apr. 20, 1933 

1. A CLEARING AGENT FOR USE IN THE PREPARATION OF HISTOLOGICAL TISSUE FOR MICROSCOPIC EXAMINATION, COMPRISING THE FOLLOWING INGREDIENTS IN ABOUT THE PROPORTIONS, BY VOLUME, OF 50% OF N. BUTYL ACETATE, 37.5% OF ETHYLENE DICHLORIDE, AND 12.5% OF CARBON TETRACHLORIDE. 